Includes bibliographical references (leaves 109-130). / Although the incidence and mortality due to the oesophageal squamous cell carcinoma (OSCC) in Black South Africans is extremely high, very little is known about the aetiology and molecular biology of the disease. In order to make a contribution to the understanding to the causes of this disease we investigated the role of the polymorphisms in the genes coding for the cytochrome P450 (CYP1A1, CYP1A2, CYP1B1, CYP2E1), sulphotransferase 1A1 (SULT1A1), glutathione S-transferases (GSTT1. GSTM1 and GSTP1) alcohol dehydrogenase (ADH2 and ADH3) and aldehyde dehydrogenase (ALDH2) because the products of these genes are involved in the metabolism or biotransformation of harmful compounds.

Includes bibliographical references. / Current chemotherapies for oesophageal cancer display poor efficacy and tolerability, highlighting an unmet need for novel chemotherapeutic agents. Artemisinin derivatives, currently used to treat malaria, were recently shown to possess potent anticancer activity. This study investigated the potential of two first generation artemisinin derivatives (artesunate and dihydroartemisinin), together with novel artemisinin hybrid compounds, as cancer chemotherapeutic agents and explored the mechanism of action in oesophageal cancer. Artesunate and dihydroartemisinin including seventeen other artemisinin derivatives were screened against oesophageal cancer cells using the 3 - [4,5-dimethylthiazol-2 -yl]-2,5 - diphenyltetrazolium bromide (MTT) assay and GraphPad Prism Software to calculate IC 50 (50% inhibitory concentration) values. Novel halogenated artemisinin - isatin hybrid compounds displayed the best activity against oesophageal cancer cells, and were more potent than artesunate and dihydroartemisinin in a small panel of oesophageal, breast and cervical cancer cell lines tested. The novel derivatives induced a G0/ G1 cell cycle arrest whilst the parental compounds induced a G2/ M block of the cell cycle, using flow cytometry. This suggested a different mechanism of action for the novel compounds. Dihydroartemisinin and the most active novel hybrid, EXP57EA, were investigated to understand their molecular mechanisms of action in oesophageal cancer.

Computer-aided drug design has become a promising alternative to high-throughput screening by identifying potential hits in silico for in vitro evaluation. In this study a combination of ligand-based and structure-based virtual screening was performed to identify in silico hits. This was based on finding similar inhibitors to 6-amino-4-(4-phenoxyphenylethylamino) quinazoline, a potent inhibitor of the Nuclear Factor kappa B (NF-κB), a transcription factor that has a pivotal role in cancer survival and Pentamidine, an anti-parasitic drug that has recently been demonstrated to possess tumour-killing activity. A hierarchical methodology consisting of a similarity search followed by structure-based virtual screening of the ZINC database was performed. In order to perform the docking studies, binding sites for 6-amino-4-(4-phenoxyphenylethylamino) quinazoline on the NF-κB/IκBα complex were identified through blind docking. In addition, the National Cancer Institute (NCI) database was screened, utilising existing structure-activity relationship data from literature. A pharmacophore search was designed to test the hypothesis of the structural features necessary for activity as seen with quinazoline inhibitors of NF-κB. No virtual hits from the ZINC database were confirmed with in vitro activity. On the other hand, three compounds identified from the pharmacophore search were confirmed to inhibit cancer cell proliferation in vitro, with compound NSC727152 demonstrating the most potent activity. In order to determine if NSC727152 acted similarly to 6-amino-4-(4-phenoxyphenylethylamino) quinazoline by inhibiting NF-κB, the effects of NSC727152 on the expression of NF-κB targeted genes, including the Growth Arrest and DNA Damage 45 (GADD45) α and γ and the Interleukin 6 (IL-6) genes were evaluated. GADD45 α and γ have been shown to be regulated by NF-κB during cancer progression and aberrant IL-6 gene expression has been implicated in cancer progression and mortality and its expression is at least partially mediated via constitutive activation of NF-κB. In this study, it has been demonstrated that GADD45 α and γ are upregulated after treatment with NSC727152. A down-regulation of the IL-6 promoter activity and mRNA expression in cancer cells treated with NSC727152 has also been demonstrated in this study. However, no hits similar to Pentamidine were confirmed with in vitro activity. In conclusion, the compound NSC727152 has been shown to inhibit NF-κB and further analysis is necessary to determine its full potential as an NF-κB inhibitor.

Bibliography: leaves 123-146. / The hypothalamic neuropeptide, gonadotropin releasing hormone (GnRH) perferentially inteacts with GnRH type I receptors on the gonadotropes in the anterior pituitary. Activation of the GnRH receptor is required for the biosynthesis and release of luteinizing hormone (LH) and follicle stimulating hormone (FSH), which regulate reproductive function. Multiple forms of GnRH are present in most vertebrate species and are thought to have physiological functions in addition to regulating pituitary hormone release. The mammalian type I GnRH receptor is proposed to discriminate between endogenous forms of GnRH (King and Millar, 1995). In this thesis the mechanism of the GnRH selectivity by a mammalian type I GnRH receptor is examined at molecular level and previous hypotheses are re-evaluated.

Includes bibliographical references (p. 100-119). / Four mutations of the CCR5 receptor have been identified in the South African population, but the effects of these mutations on CCR5 function and HIV infection are unknown. We have used in vitro methods to assess the ffect of the mutations, Asp2Val, Leu107Phe, Arg225Gln and Arg225stop, on CCR5 interactions with chemokine ligands and HIV.

Bibliography: p. 207-213.

One of the major focus areas of anticancer therapy is the design of new radiotherapeutic agents that are able to specifically target and destroy cancer cells with minimal side effects and damage to healthy, normal cells. This thesis describes studies towards the synthesis of a macromolecular bioconjugate that was designed to: i) co-ordinate a radioisotope through a tetra-amine macrocycle (cyclam), ii) lead to passive tumour targeting via the EPR effect and a suitably large carrier such as human serum albumin and iii) induce active targeting through a glucose moiety recognised by the over-expressed glucose transporters on the surface of highly metabolically active cancer cells. The various cyclam functionalisation strategies explored were relatively unsuccessful, but eventually a bis-aminal cyclam was successfully converted, through nucleophilic substitution, into a precursor pro-conjugate: a di-functionalised cyclam containing a β-glycoside tether and a long chain primary alkylamine. The glycoside tether was synthesised via glycosylation of a glycosyl iodide with decandiol followed by oxidation of the terminal hydroxyl group to an acid chloride for cyclam acylation. The second linker attached to cyclam was synthesised by conversion of decanediol to a brominated alkyl amine. This amine would then be converted into a maleimide functionality suitable for Michael addition with a free thiol group contained within the proposed bio-carrier to form the desired bioconjugate. Further studies described towards the synthetic construction of the bioconjugate include: 1) The construction of a maleimide group 2) The attachment of an imaging radioisotope, ⠹⠹m Tc, or therapeutic isotope, ¹⠰³ Pd, to the pro- conjugate and other glucose-cyclam precursors 3) The determination of the potential uptake of the bioconjugate through glucose transporters by using a fluorescent dansyl-glucose compound as a model and monitoring its uptake into WHCO1 oesophageal cancer cells. 4) The HPLC analysis of the coupling of a glucose-maleimide model compound to bovine serum albumin to investigate the Michael addition of the free thiol in HSA to a maleimide 5) The development of a potentially alternative nanoparticle carrier by synthesis of palladium and magnetic nanoparticles with commercially available thioglucose or glucuronic acid moieties as the surface targeting and stabilising agent. In summary, this thesis outlines a number of synthetic, radiological and biological aspects towards the development of a fully functioning radiolabelled macromolecular bioconjugate that could be tested for improved targeted cancer radiotherapy.

Sporadic colorectal cancer (CRC) has been linked to various lifestyle factors, including the consumption of alcohol and red meat, smoking, and obesity. CRC is one of most extensively characterised cancers, both at a molecular and 'omic' level; nevertheless, the precise mechanism driving CRC initiation remains unknown. To date, numerous studies have identified changes in the microbial profiles of CRCs compared to adjacent normal mucosa and compared to healthy controls; however, CRC-associated bacteria have not been concurrently quantified across a single cohort; nor have the relationships between CRC-associated bacteria, clinicopathological features of CRC and genomic subtypes of CRC been investigated. The main aim of this thesis was therefore to gain insight into the potential contribution of CRC-associated bacteria in the aetiopathogenesis of CRC by leveraging both host genomic and clinicopathological data as well as to investigate patterns of tissue colonisation between different CRC-associated bacteria. The objectives were 1) to quantify, using quantitative-PCR, CRC-associated bacteria in a cohort of 55 paired tumour and adjacent histologically normal samples collected during surgical resection as well as in an additional 18 formalin-fixed paraffin-embedded (FFPE) samples; 2) to determine their relationships to patient age, gender, ethnicity, stage of disease, site of disease and MSI status (Chapter 4); 3) to evaluate the relationship between each bacterium and host gene expression (Chapter 8) and methylation changes (Chapter 6); and 4) to determine genomic subtypes of CRC using unsupervised clustering of gene expression data in the context of patient clinicopathological features and bacterial quantitation data; and 5) to gain a deeper biological understanding of the results from the objectives 1–4 using pathway analyses of the genomic subtypes obtained (Chapter 7). The main finding of this thesis is that a transcriptomic subtype of colorectal cancer, characterised by an increase in CpG island methylation, displays an increased frequency of colonisation by Enterococcus faecalis and by high levels of Fusobacterium. At the pathway-level, this subtype is enriched for pathways related to damage response, infection, inflammation and cellular proliferation; notably, these findings were confirmed in a well-defined publically available CRC gene expression dataset of colorectal adenocarcinomas (N=155). These findings suggest that specific bacterial colonisation underlie s a distinct genomic subtype of colorectal cancer that is characterise d by inflammatory-related gene expression changes ; these findings however require validation in a larger cohort. In addition, novel associations between colonisation by specific bacteria and host clinicopathological, transcriptomic and DNA methylation features were identified.

Includes abstract. / Includes bibliographical references. / Oesophageal squamous cell carcinoma (OSCC) is the 8th most common cancer worldwide with high incidence in areas that include China, Iran and South Africa. The current treatment available for OSCC does not significantly enhance patient survival. A better understanding of proliferative and survival pathways activated in OSCC could allow identification of more specific therapeutic targets, potentially improving management of OSCC. Cell surface receptors are known to play important roles in relaying signals from the extracellular environment...The aim of this study was to determine the role of EGFR, IGF-1R and CXCR2 in proliferation and survival of OSCC cells.

In this study, novel disulphide bridges were engineered into the linker region of ACE [Angiotensin-converting enzyme] in an attempt to limit inter-domain movement, thereby producing a candidate for crystallisation and to determine the effect of these bridges on inter-domain movement.